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1.
Fig. 6.

Fig. 6. From: Overexpression of Hepatocyte Nuclear Factor-4? Initiates Cell Cycle Entry, but Is not Sufficient to Promote ?-Cell Expansion in Human Islets.

Overexpression of Cyclin D3 and Cdk6 activates the DNA damage response in human β-cells. Dual immunostaining for γH2AX (green), DAPI (blue), and (A) Pdx1 (red), (B) insulin (red), (D) BrdU (red), and (E) Ki67 (red) in Cyclin D3 and Cdk6-transduced human islets. C, Quantification of the percentage of either Pdx1+, or insulin+ cells that are γH2AX+ in primary human islets transduced with both Cdk6 and Cyclin D3 (n = 3–4 per group). F, Quantification of the percentage of either BrdU+,total, BrdU+,diffuse, BrdU+,punctate, Ki67+,total, Ki67+,normal, or Ki67+,irregular cells that are γH2AX+ in primary human islets transduced with both Cdk6 and Cyclin D3 (n = 3–4 per group). G, Quantification of the percentage of γH2AX+ cells that are either BrdU+,diffuse, BrdU+,punctate, Ki67+,normal, or Ki67+, irregular (n = 3–4 per group; *, P < 0.001 BrdU+,punctate vs. Ki67+,irregular populations). H, Immunostaining for phospho-p53 on serine 15 (green), DAPI (blue), and BrdU (red) in Cyclin D3 and Cdk6-transduced human islets. I, Quantification of the percentage of either BrdU+,diffuse or BrdU+,punctate that are phospho-p53 (serine 15)+ (n = 4; *, P < 0.001). The white arrowheads indicate colocalization, and yellow arrowheads indicate noncolocalization between two markers. All human islets were incubated for 72 h after transduction. The scale bars indicate 25 μm in panels A and H and 5 μm in the insets of panels E and H. p, Punctuate; d, diffuse; n, normal; i, irregular.

Sebastian Rieck, et al. Mol Endocrinol. 2012 September;26(9):1590-1602.
2.
Fig. 4.

Fig. 4. From: Overexpression of Hepatocyte Nuclear Factor-4? Initiates Cell Cycle Entry, but Is not Sufficient to Promote ?-Cell Expansion in Human Islets.

Partial loss of lineage fidelity is the predominant fate of β-cells overexpressing HNF4α8. Immunofluorescence analysis of Pdx1 (green), TUNEL (red), and DAPI (blue) in (A) untransduced, (B) AdCMV-eGFP-, and (C) AdRIP-hHNF4α8-transduced islets at 72-h time point. D, Quantification of the percentage of Pdx1+ cells that are TUNEL+ in each experimental condition 72 h after transduction (n = 3–4 for each group). Immunostaining for HNF4α (green), TUNEL (red), and DAPI (blue) in AdRIP-hHNF4α8-transduced islets at (E) 72-h, and (F) 120-h time points. G, Quantification of the percentage of HNF4High cells that are Tunel+ at 72 and 120 h after transduction (n = 3 per time point). Analysis of TUNEL (red) positive cells that are BrdU+ (green) at (H) 72 h and (I) 120 h after transduction with AdRIP-hHNF4α8. J, Quantification of the percentage of either BrdU+,diffuse or BrdU+,punctate cells that are TUNEL+ at 72 h and 120 h (n = 3–5 per time point). Immunodetection of Nkx6.1 (green), BrdU (red), and DAPI (blue) in HNF4α8 transduced islets (K) 48 h and (L) 120 h after transduction. M, Quantification of the percentage of BrdU+,punctate cells that are Nkx6.1+ at 48 and 120 h (*, P < 0.001; n = 3 per time point). Immunodetection of MafA (green), BrdU (red), and DAPI (blue) in HNF4α8-transduced islets (N) 48 h and (O) 120 h after transduction. P, Quantification of the percentage of BrdU+,punctate cells that are MafA+ at 48 and 120 h (n = 2–3 per time point). The yellow arrows indicate BrdU+,punctate, Nkx6.1, or MafA cells, and white arrows highlight their colocalization. The scale bars in panels C, F, H, and O indicate 25 μm. n.s., P = 0.28.

Sebastian Rieck, et al. Mol Endocrinol. 2012 September;26(9):1590-1602.
3.
Fig. 5.

Fig. 5. From: Overexpression of Hepatocyte Nuclear Factor-4? Initiates Cell Cycle Entry, but Is not Sufficient to Promote ?-Cell Expansion in Human Islets.

HNF4α8 synergizes with factors known to be sufficient for promoting cell cycle entry in human β-cells. Immunofluorescence detection of Ki67 (green), Pdx1 (red), and DAPI (blue) in (A) Cyclin D3 and Cdk6-, and (B) HNF4α, Cyclin D3, Cdk6-transduced primary human islets at 72 h with (C, left graph) quantification of the percentage of Pdx1+ cells that are Ki67+,total (*, P < 0.04; n = 6 per group). Image (A) is shown as an example of normal Ki67 staining pattern in Cyclin D3/Cdk6 transduction cocktail. C (right graph), Quantification of the percentage of Pdx1+ cells that are either Ki67+,normal or Ki67+,irregular in Cdk6/CyclinD3 and Cdk6/Cyclin D3/HNF4α8 conditions (n = 6 per group). Immunofluorescent detection of HNF4α (red), DAPI (blue), and (D) Ki67, (E) Cyclin A in HNF4α8, Cyclin D3, Cdk6-transduced primary human islets at 72 h with quantification of the percentage of HNF4αHigh cells that are (F) either Ki67+,normal or Ki67+,irregular, and (G) CyclinA+ (n = 3 per group). Immunostaining of HNF4α (red), DAPI (blue), and (H) γH2AX (green), (I) TUNEL (red) in HNF4α8, Cyclin D3, Cdk6-transduced islets at 72 h with quantification of the percentage of HNF4αHigh cells that are (J) γH2AX+ and (K) TUNEL+ (n = 3 per group). The inset in panel I shows representative DAPI staining (blue) of HNF4AHigh TUNEL+ cells. The scale bars in panels B, D, and I indicate 25 μm and in inset of I indicates 5 μm. n, Normal; I, irregular.

Sebastian Rieck, et al. Mol Endocrinol. 2012 September;26(9):1590-1602.
4.
Fig. 7.

Fig. 7. From: Overexpression of Hepatocyte Nuclear Factor-4? Initiates Cell Cycle Entry, but Is not Sufficient to Promote ?-Cell Expansion in Human Islets.

Cell cycle progression of islet cells can be discerned by the manner of thymidine analog incorporation and multiple marker analysis. Schematic illustrating the cell cycle characteristics of cell types present in an HNF4α8-transduced human adult islet in vitro. Left column, Nonendocrine islet cells successfully turn over, distinguished by proper thymidine incorporation (diffuse), and activation of multiple cell cycle proteins. Middle column, Conversely, human adult β-cells are almost all quiescent and do not attempt cell cycle entry. The majority of HNF4α8-overexpressing cells do not show any attempt at cell cycle entry and progression. Right column, However, some of the HNF4αHigh β-cells are able to initiate cell cycle entry but activate cell cycle arrest programs such as the DNA damage response in an attempt to fix an apparent S-phase insult (exemplified by punctate thymidine analog domains). Asterisk, CyclinD3/Cdk6- and CyclinD3/Cdk6/HNF4α8-overexpressing β-cells fall into the latter category as they do show signs of active cell cycling and DNA damage response. However, the successful ability of these cycling β-cells to halt cell cycle progression upon double stranded DNA damage might vary depending on the cocktail of genes being constitutively overexpressed.

Sebastian Rieck, et al. Mol Endocrinol. 2012 September;26(9):1590-1602.
5.

Fig. 2. From: Overexpression of Hepatocyte Nuclear Factor-4? Initiates Cell Cycle Entry, but Is not Sufficient to Promote ?-Cell Expansion in Human Islets.

HNF4αHigh β-cells arrest in the cell cycle. Analysis of Ki67 (green), BrdU (red), and DAPI (blue) cells in primary human islets at (A) 24 h, (B) 42 h, and (C) 72 h after transduction with AdRIP-hHNF4α8. D, Quantification of the percentage of BrdU+,Diffuse or BrdU+,Punctate that are Ki67+ at the 24-, 30-, 36-, 42-, 48-, and 72-h time point (72 h, BrdU+,diffuse group is statistically significantly lower then 24, 30, 36, 42 h, BrdU+,diffuse groups, *, P < 0.01; 48 h, BrdU+,diffuse groups are statistically lower then 24-h BrdU+,diffuse group; *, P < 0.01; both determined by one-way ANOVA with Tukey post hoc test; n = 3–5 for each type of BrdU incorporation per time point). E and F, Immunostaining of Cyclin A (green) with BrdU (red) in human islets 42 h after transduction with AdRIP-hHNF4α8. G, Quantification of the percentage of BrdU+,Diffuse or BrdU+,Punctate that are Cyclin A+ (*, P < 0.01; n = 3 per type of BrdU incorporation). H and I, Immunostaining of Mcm7 (green) with BrdU (red) in human islets 42 h after transduction with AdRIP-hHNF4α8. J, Quantification of the percentage of BrdU+,Diffuse or BrdU+,Punctate that are Mcm7+ (*, P < 0.01; n = 3 per type of BrdU incorporation). K and L, Immunostaining of phospho-p53(ser15) (green) and BrdU (red), and (M and N) phospho-Chk2 (green) and BrdU (red) in human islets 48 h after transduction with AdRIP-hHNF4α8. O, Quantification of the percentage of BrdU+,Diffuse or BrdU+,Punctate that are either phospho-p53(Ser15)+ or phospho-Chk2+ (*, P < 0.005; n = 3 per type of BrdU incorporation and protein examined). Immunofluorescence of p53 (green) and BrdU (red) in human islets (P) 48 h and (Q) 72 h after transduction with AdRIP-hHNF4α8. R, Quantification of the percentage of BrdU+,punctate cells that are p53+ at 48-h and 72-h time points (n = 3 per time point). Immunostaining of p16 (green) and BrdU (red) in human islets (S) 48 h and (T) 120 h after transduction with AdRIP-hHNF4α8. White arrows indicate either diffuse (d) or punctate (p) BrdU incorporation. The scale bars in panels C and T indicate 25 μm. n.s., P = 0.30.

Sebastian Rieck, et al. Mol Endocrinol. 2012 September;26(9):1590-1602.
6.

Fig. 1. From: Overexpression of Hepatocyte Nuclear Factor-4? Initiates Cell Cycle Entry, but Is not Sufficient to Promote ?-Cell Expansion in Human Islets.

HNF4αHigh β-cells incorporate BrdU in a punctate, not diffuse, manner. Immunofluorescence staining of Pdx1 (green), BrdU (red), and DAPI (blue) in primary human islets (A) not transduced with adenovirus, (B) transduced with AdCMV-eGFP, and (C) transduced with AdRIP-hHNF4α8. C (inset), An example of a nucleus exhibiting punctate BrdU incorporation (red) overlapping with DAPI-dimished regions (blue). Immunofluorescence staining of punctate BrdU incorporation (red) and DAPI (blue) with (D) insulin (green), (E) glucagon (green), and (F) somatostatin (green) cells in primary human islets overexpressing HNF4α8. Similarly, detection of diffuse BrdU incorporation (red) and DAPI (blue) with (G) insulin (green), (H) glucagon (green), and (I) somatostatin (green) cells in AdRIP-hHNF4α8-transduced human islets. I (inset), An example of two DAPI-stained (blue) nuclei showing diffuse BrdU incorporation (red). J, Quantification of the percentage of Pdx1+ cells that are either BrdU+,diffuse or BrdU+,punctate in untransduced, AdCMV-eGFP, and AdRIP-hHNF4α8-transduced islets (HNF4α8, BrdU+,punctate group is statistically significantly higher compared with all other groups; *, P < 0.001 as determined by one-way ANOVA with Tukey post hoc test; n = 3 for each group). K, Quantification of the percentage of insulin+, glucagon+, or somataostatin+ cells that are either BrdU+,diffuse or BrdU+,punctate in AdRIP-hHNF4α8-transduced islets (insulin+, BrdU+,punctate group is statistically significantly higher compared with all other groups; *, P < 0.001 as determined by one-tailed ANOVA with Tukey post hoc test; n = 3–4 for each hormone+ population). Detection of HNF4α (green), BrdU (red), and DAPI (blue) in primary human islets (L) not transduced with adenovirus, (M) transduced with AdCMV-eGFP, and (N) transduced with AdRIP-hHNF4α8. O, Quantification of the percentage of either BrdU+,diffuse or BrdU+,punctate cells that are HNF4αHigh in AdRIP-hHNF4α8-transduced islets. (*, P < 0.001; n = 4 each BrdU incorporation pattern). P, Quantification of the percentage of HNF4αHigh cells are BrdU+,punctate in nondiabetic or type 2 diabetic human islet donations transduced with in AdRIP-hHNF4α8 (n = 3–4 for each diabetic status). All primary human islets were harvested 72 h after transduction. White arrows indicate either diffuse (d) or punctate (p) BrdU incorporation. The scale bar in panels I and N indicates 25 μm. GIUC, glucagon; INS, insulin; SOMATO, somatostatin. n.s., P = 0.28.

Sebastian Rieck, et al. Mol Endocrinol. 2012 September;26(9):1590-1602.
7.

Fig. 3. From: Overexpression of Hepatocyte Nuclear Factor-4? Initiates Cell Cycle Entry, but Is not Sufficient to Promote ?-Cell Expansion in Human Islets.

Overexpression of HNF4α8 leads to activation of the DNA damage response associated with replication stress. Immunodetection of γH2AX (green), Pdx1 (red), and DAPI (blue) in untransduced (panel A), GFP- (panel B), and HNF4α8-overexpressing (panel C) primary human islets at 72 h after transduction. The inset in panel B shows GFP (red), γH2AX (green), and DAPI (blue) colocalization. D, Quantification of the percentage of Pdx1+ cells that are γH2AX+ in all experimental conditions (AdRIP-hHNF4α8 group is statistically significantly higher then untransduced and AdCMV-eGFP groups; *, P < 0.03 as determined by one-way ANOVA with Tukey post hoc test; n = 3 for each group). Immunolocalization of γH2AX (green), and both (panels E and F) HNF4α (red) and (panels G and H) BrdU (red) in HNF4α8-tranduced primary human islets at (panels E and G) 72 h and (panels F and H) 120 h after transduction. G (inset), Example of a BrdU+,punctate cell with enlarged nucleus at 72-h time point. I, Quantification of the percentage of HNF4αHigh cells that are γH2AX+ at 72 and 120 h after AdRIP-hHNF4α8 transduction (n = 3–4 per time point). J, Quantification of the percentage of either BrdU+,diffuse or BrdU+,punctate cells that are γH2AX+ at 72 h and 120 h (n = 3; *, P < 0.001 vs. BrdU+,Diffuse condition for each time point). Simultaneous immunodetection of EdU (red) and BrdU (green) in HNF4α8-transduced islets of (K) single thymidine analog-labeled diffuse nucleus with 1-h nonlabeling time, (L) dual labeled noncolocalizing diffuse nucleus with 1-h labeling time, dual-labeled colocalizing punctate nucleus with (M) 1 h, (N) 3 h, and (O) 6 h nonlabeling times. Individual red channels (K′–O′), and green channels (K″–O″) are shown. The white arrows indicate dual-labeled replication foci. The scale bars in panels C and H indicate 25 μm and in panel O indicate 5 μm, respectively. n.s., P = 0.29.

Sebastian Rieck, et al. Mol Endocrinol. 2012 September;26(9):1590-1602.

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